T Cells and their Functions:
T cells are lymphocytes that have receptors capable of recognizing protein fragments (antigens) derived from foreign, potentially harmful proteins or organisms such as bacteria and viruses or from proteins present in the body of the host. Each T cell receptor recognizes a different string of amino acids, which comprise the antigen. Essentially there will always be at least one T cell receptor in the total repertoire of T cells, which will recognize any given antigen, which is in the body.
There are two main types of T cells, namely CD4+ helper T cells and CD8+ killer T cells. Helper T Cells (Th) carry receptors that engage antigens present on the surfaces of an antigen-presenting cell (APC) such as dendritic cells and sometimes macrophages. It is only by engagement with an antigen present on an APC and a subsequent process known as co-stimulation that a Th cell can become activated so that it may attack that specific antigen. Before the Th cell has become activated it is known as a “naïve” T cell. After the Th cell has become activated it becomes an “effector” T cell and wages an immune attack against the particular antigen. After cells containing the antigen have been destroyed, most of the effector T cells die. However, some effector T cells remain in a resting or quiescent state and are then known as “memory T cells.” At least two types of Memory T cells exist, each having different migratory characteristics and effector functions. The first type of memory T cells are known as “effector memory T cells” (TEM) and produce IFN-γ, TNF-α and IL-2 or pre-stored perforin (in the case of CD8s) when they encouner an antigen. The second type of memory T cells, known as “central memory T cells” (TCM), express the chemokine receptor CCR7 similar to naïve T cells and lack immediate effector function. When TEM cells encounter the same antigen that initially caused their activation, they quickly convert back to effector T cells without the need for co-stimulation. Such rapid redeployment of effector T cells without the need for co-stimulation allows the immune system to attack the antigen in a very efficient manner.
Ion Channels: Molecular Targets for Pharmacologic Intervention
Ion channels are proteins embedded within the cell membrane that control the selective flux of ions across the membrane, thereby allowing the rapid movement of ions during electrical signaling processes. Because ion concentrations are directly involved in the electrical activity of excitable cells (e.g., neurons), the functioning (or malfunctioning) of ion channels can substantially control the electrical properties and behavior of such cells. Indeed, a variety of disorders, broadly termed as “channelopathies,” are believed to be linked to ion channel insufficiencies or dysfunctions.
Ion channels are referred to as “gated” if they can be opened or closed. The basic types of gated ion channels include a) ligand-gated channels, b) mechanically gated channels and c) voltage-gated channels. In particular, voltage-gated channels are found in neurons and muscle cells. They open or close in response to changes in the potential differences across the plasma membrane.
In recent years, drug development efforts have included work aimed at identifying and characterizing various ion channels and designing agents that increase or decrease the flux of ions through those ion channels to bring about desired therapeutic effects.
Kv1.3 Channels and their Roll in T Cell Physiology.
The predominant voltage-gated potassium ion channel in human T-lymphocytes is encoded by Kv1.3, a Shaker-related gene. Kv1.3 channels have been characterized extensively at the molecular and physiological level and are known to play a vital role in controlling T-lymphocyte proliferation, mainly by maintaining the membrane potential of resting T-lymphocytes. For example, encephalitogenic and arthritogenic rat T cells that have been chronically activated with myelin antigens have been shown to express a unique channel phenotype (high Kv1.3 channels and low IKCa1 channels), distinct from that seen in quiescent and acutely activated T cells (Beeton et al., 2001, Selective blockade of T lymphocyte K(+) channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. USA 98:13942) and such findings have been confirmed in myelin antigen specific T cells from human patients suffering from multiple sclerosis (MS). Contrary to myelin-reactive T cells from healthy controls and to mitogen or control antigen activated T cells from MS patients, myelin reactive T cells from MS patients predominantly expressed surface markers of terminally differentiated effector memory T cells (CCR7−CD45RA−) and exhibited the Kv1.3highIKCa1low phenotype (Wulff et al., The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. 2003, J. Clin. Invest. 111:1703). In the same study, it was shown that this special K+ channel phenotype made the proliferation of effector memory T cells highly sensitive to inhibition by Kv1.3 blockers. Naïve and central memory T cells were only affected at 10-fold higher concentrations of Kv1.3 blockers and could escape Kv1.3 inhibition during subsequent stimulation through the up-regulation of the calcium-activated potassium channel IKCa1. Thus, it may be possible to develop a selective potassium channel blocker that will target the disease-inducing effector memory T cell population without affecting the normal immune response.
Kv1.3 and IKCa1 Expression and Functional Roles in Naïve and Memory T-Cells
Naïve, central memory (TCM) and effector memory T (TEM) cells are classified based on the expression of the chemokine receptor CCR7 and the phosphatase CD45RA. Naïve (CCR7+CD45RA+) and TCM (CCR7+CD45RA−) cells migrate to the lymph node using CCR7 as an entry code, before migrating to sites of inflammation. In contrast, TEM cells have the ability to home directly to sites of inflammation, where they can secrete high amounts of interferon (IFN-γ) and tumor necrosis factor-α (TNF-α) and exhibit immediate effector function. The expression patterns of Kv1.3 and IKCa1 change dramatically as naive cells become memory cells. At rest, CD4+ and CD8+ T-cells of all three subsets exhibit ˜200 to 400 Kv1.3 channels, and 0 to 30 IKCa1 channels (Wulff et al., The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. 2003, J. Clin. Invest. 111:1703). Activation has diametrically opposite effects on channel expression; as naive and TCM cells move from resting to proliferating blast cells, they transcriptionally up-regulate IKCa1 to ˜500 channels per cell. In contrast, activation of TEM cells enhances Kv1.3 expression without any change in IKCa1 levels (Wulff et al., 2003, J. Clin. Invest. 111:1703). Functional Kv1.3 expression increases dramatically within 15 h of activation to a level of 1500 Kv1.3 channels/cell, remains elevated for the following 48 to 72 h, and then returns to baseline over the next five days (Beeton et al., A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes. 2003, J. Biol. Chem. 278:9928)
The subset-specific channel expression has important functional consequences, since Kv1.3 and IKCa1 regulate Ca2+ entry into T-cells through Ca2+-release-activated Ca2+ channels that exhibit ‘upside-down’ voltage-dependence compared with voltage-gated Ca2+ channels. A negative membrane potential drives Ca2+ entry through these channels. The electrochemical gradient supporting Ca2+ entry is initially large, resulting in significant Ca2+ influx. However, Ca2+ entry results in depolarization of the plasma membrane, limiting further influx. To maintain Ca2+ entry over the time scale required for gene transcription, a balancing cation efflux is necessary; this is provided by the efflux of K+ ions through Kv1.3 and/or IKCa1 channels, which supply the electrochemical driving force for Ca2+ entry via membrane hyperpolarization.
Depolarization resulting from Kv1.3 and IKCa1 blockade is inhibitory for Ca2+ influx, signaling and lymphocyte activation. As Kv1.3 channels predominate in resting T-cells of the three subsets, the Kv1.3 blocker ShK, but not the IKCa1 blocker TRAM-34, suppress antigen or mitogen-driven activation. However, ShK is 10-fold more effective on TEM cells than on naive and TCM cells (IC50 values of 400 pM and 4 nM, respectively), due to the fact that the latter cells rapidly up-regulate lkCa1 after stimulation and become less sensitive to Kv1.3 inhibitors (Wulff et al., The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. 2003, J. Clin. Invest. 111:1703). Once IKCa1 is up-regulated in naïve and TCM cells, the reactivation of these cells is sensitive to IKCa1 but not Kv1.3 blockade. Naïve and TCM cells can up-regulate IKCa1 following mitogen or antigen stimulation, even if their initial activation is suppressed by Kv1.3 blockade; and can consequently escape further inhibition by Kv1.3 inhibitors (Wulff et al., 2003, J. Clin. Invest. 111:1703). Early in vivo studies support these in vitro findings. The Kv1.3 blockers MgTX (Koo et al., Blockade of the voltage-gated potassium channel Kv1.3 inhibits immune responses in vivo. 1997, J. Immunol. 158:1520) and correolide (Koo et al., Correolide and derivatives are novel immunosuppressants blocking the lymphocyte Kv1.3 potassium channels. 1999, Cell Immunol. 197:99) effectively suppress the primary delayed-type hypersensitivity (DTH) response in mini-pigs, but are much less effective in suppressing the secondary DTH response, presumably due to the fact that the activated naïve or TCM cells involved have up-regulated IKCa1 expression. In contrast, TEM cells exclusively up-regulate Kv1.3 channels, and are persistently suppressed by Kv1.3 inhibitors.
Kv1.3 and IKCa1 Expression and Functional Roles in Naïve and Memory B-Cells
A similar change in potassium channel expression takes place during the differentiation from naïve into class-switched memory B cells. While naïve (IgD+CD27−) and “early” memory B cells (IgD+CD27+) rely on IKCa1 for their proliferation, class-switched (IgD−CD27+) memory B cells rely on Kv1.3 and their proliferation is therefore potently inhibited by the Kv1.3 blockers ShK and Psora-4 (Wulff et al. K+ channel expression during B cell differentiation: implications for immunomodulation and autoimmunity. 2004. J. Immunol. 173:776-86). Thus, Kv1.3 blockers selectively target “late” memory responses in both the T- and B-cell lineage should be useful for the treatment of autoimmune disorders.
Kv1.5 Channels and Regulation/Deregulation of Cardiac Rhythm
Ion flux through voltage gated potassium channels also plays a role in regulation of cardiac rhythms. Atrial fibrillation (AF) is a common cardiac rhythm disturbance. AF can be treated or prevented by agents that prolong the atrial action potential duration and refractoriness. Indeed, drugs such as dofetilide, almokalant, amiodarone and d-sotalol can effectively suppress AF. However, such drugs may also prolong the ventricular action potential duration, thereby giving rise to life threatening or lethal ventricular arrhythmias. This potential for antiarrhythmic drugs to actually cause certain types of arrhythmias while preventing others is sometimes referred to as the drug's “proarrhythmic potential.” Proarrhythmic potential is an important dose-limiting factor in the use of antiarrhythmic drugs. In fact, a common proarrhythmic event reported to result from the use of traditional antiarrhythmic drugs that prolong ventricular repolarization (QT interval) to treat AF is a condition known as torsades de pointes, which is a rapid polymorphic ventricular tachycardia.
Because voltage gated Kv1.5 potassium channels are predominantly located in atrial tissue, drugs that inhibit Kv1.5 channels are being developed for the treatment of AF (Brendel, J. and Peukert, S.; Blockers of the Kv1.5 Channel for the Treatment of Atrial Arrhythmias; Current Medicinal Chemistry—Cardiovascular & Hematological Agents, Volume 1, No. 3, 273-287 (2003)). Drugs that selectively inhibit Kv1.5 channels could prove to be a viable new approach for the treatment of AF with minimal or no proarrhythmic potential. However, it is also possible that, untoward inhibition of Kv1.5 channels in patients who have normal heart rhythms could induce an electrical imbalance and actually cause arrhythmias in such patients. Thus, when developing drugs that are intended to inhibit potassium channels other than Kv1.5 (e.g., drugs intended to inhibit Kv1.3 channels to treat T cell mediated diseases), it may be desirable to design these drugs to display selectivity for the target potassium channels (e.g., Kv1.3 channels) over the heart-affecting Kv1.5 channels.
In view of the foregoing, there remains a need for the synthesis and development of new potassium channel inhibitors that are specific for certain potassium channels over other potassium channels, thereby providing specific therapeutic effects with minimal side effects.